The long term goals of this research project are: 1) the isolation and in vitro construction of Sindbis virus mutants which differ in their pathogenic effects on experimental animals, 2) the detailed characterization of such mutants as to genome sequence and virion topography and correlation of specific mutations with their biological and pathogenesis phenotypes, and 3) the use of this model system to explore informed molecular approaches to the development of live alphavirus vaccines. The ability to construct in vitro recombinant viruses and generate site-directed mutations is one of two considerations which make the Sindbis system an excellent model for the detailed examination of the molecular basis of alphavirus neurovirulence. The second consideration is that the prior mapping of pathogenesis loci in biologically selected mutants by our group will guide the targeting of site-directed mutagenesis to specific regions. At this time, few other virus systems have both of these elements. The experiments proposed in this application will explore the molecular genetics of Sindbis virus pathogenesis in mice. Specific loci which affect pathogenesis will be mapped and characterized utilizing closely related, biologically selected mutants as well as site-directed mutagenesis of a full- length cDNA clone of the viral genome. The effects of these mutations will be assessed in animal models and in cell culture systems which display inducible differentiation toward neuron-like cells. Knowledge of the molecular genetics of Sindbis pathogenesis will be extended by the informed design of a low-reversion, model vaccine containing multiple, independently attenuating mutations. Finally, while the focus of the proposed experiments is the molecular genetics of alphavirus pathogenesis, the results obtained will be important to the understanding of penetration, virus maturation and structure, and post-translational processing by proteolytic cleavage. The proposed investigation will yield significant new information regarding the pathogenesis and replication of the alphavirus group.